Discovery and characterization of conserved binding of eIF4E1 (CBE1), a eukaryotic translation initiation factor 4E– bindingplant proteinReceived for publication, May 31, 2018, and in revised form, August 8, 2018 Published, Papers in Press, September 13, 2018, DOI 10.1074/jbc.RA118.003945
Ryan M. Patrick, Jessica C. H. Lee, Jade R. J. Teetsel, Soo-Hyun Yang, Grace S. Choy, and Karen S. Browning1
From the Department of Molecular Biosciences and Institute for Cell and Molecular Biology, The University of Texas at Austin,Austin, Texas 78712
Edited by Joseph M. Jez
In many eukaryotes, translation initiation is regulated by pro-teins that bind to the mRNA cap– binding protein eukaryotictranslation initiation factor 4E (eIF4E). These proteins com-monly prevent association of eIF4E with eIF4G or form repres-sive messenger ribonucleoproteins that exclude the translationmachinery. Such gene-regulatory mechanisms in plants, andeven the presence of eIF4E-interacting proteins other thaneIF4G (and the plant-specific isoform eIFiso4G, which bindseIFiso4E), are unknown. Here, we report the discovery of aplant-specific protein, conserved binding of eIF4E 1 (CBE1). Wefound that CBE1 has an evolutionarily conserved eIF4E-bindingmotif in its N-terminal domain and binds eIF4E or eIFiso4E invitro. CBE1 thereby forms cap-binding complexes and is aneIF4E-dependent constituent of these complexes in vivo. Ofnote, plant mutants lacking CBE1 exhibited dysregulation of cellcycle–related transcripts and accumulated higher levels ofmRNAs encoding proteins involved in mitosis than did WTplants. Our findings indicate that CBE1 is a plant protein thatcan form mRNA cap– binding complexes having the potentialfor regulating gene expression. Because mammalian translationfactors are known regulators of cell cycle progression, we pro-pose that CBE1 may represent such first translation factor–associated plant-specific cell cycle regulator.
In eukaryotes, mRNAs are co-transcriptionally capped with a5� to 5� reverse-linked 7-methylguanosine residue; the presenceof the cap structure is an important determinant of down-stream processing and regulatory fate (1). The cap moiety isrecognized by a class of proteins called cap-binding proteinswhich notably includes eukaryotic initiation factor 4E (eIF4E).2eIF4E is in turn bound by eIF4G, a large scaffolding protein, toform the eIF4F complex which has important roles in transla-tion initiation. eIF4G contacts eIF4B and the RNA helicaseeIF4A which promote unwinding of secondary structure in the
mRNA during translation initiation (2, 3). eIF4G recruits the43S pre-initiation complex to mRNA through contacts witheIF3 and eIF5. The importance of the eIF4F complex is suchthat mRNA cap recognition by eIF4E is considered a centralcontrol point in translation initiation and its regulation (4).
eIF4G contains a well-conserved motif, (Y(x)4L�), where xis any amino acid and � is a hydrophobic residue, which isrequired for eIF4E binding (5). This motif is shared by a numberof other eIF4E-binding proteins (4E-BPs) (6). In mammals,4E-BPs are regulated by phosphorylation through the mTORpathway. In the absence of phosphorylation, 4E-BPs competewith eIF4G for binding of eIF4E, sequestering the protein awayto reduce levels of translation. Activation of mTOR leads tophosphorylation of 4E-BPs, reducing their affinity for eIF4Eand making the protein available for binding by eIF4G, increas-ing levels of translation initiation (7). To date, there is no evi-dence of a similar 4E-BP-like mechanism in regulation of planttranslation (8).
Other eIF4E-binding proteins are known to contribute to arange of regulatory processes. 4E-transporter in mammals hasan important role in mRNA localization by directing nucleocy-toplasmic shuttling of eIF4E and also localizes eIF4E to P-bod-ies (9). Other types of eIF4E-binding proteins target specificmRNAs for translation repression by binding specific sequencemotifs internal to the mRNA and binding the eIF4E present atthe mRNA’s cap, effectively rendering it closed to translationalactivation. Examples of such activity are found in mammalianMaskin and Neuroguidin, eIF4E-binding proteins which inturn bind the RNA-binding protein cytoplasmic polyadenyl-ation element-binding protein (10, 11). Similarly, in Drosoph-ila, Cup binds eIF4E and RNA-binding proteins such as Smaugand Bruno which recognize specific 3� UTR elements inmRNAs targeted for translational repression (12, 13). AnotherDrosophila protein, Mextli, is expressed in ovarian germ linestem cells and binds eIF4E to promote translation in a mannerapparently independent of eIF4G (14).
Flowering plants, in addition to the conserved eIF4E andeIF4G, have the plant-specific isoforms eIFiso4E and eIFiso4Gwhich make up a complex called eIFiso4F (2). The exact mech-anisms by which translation initiation is regulated by eIF4Fand/or eIFiso4F in plants remain unclear by comparison tomammals and fungi. As no proteins similar to 4E-BP have beenidentified, and binding affinity between eIF4E/eIF4G andeIFiso4E/eIFiso4G is significantly higher than other eukaryotic
This work was supported by National Science Foundation Grant MCB1052530(to K. S. B.). The authors declare that they have no conflicts of interest withthe contents of this article.
RNA sequences generated in this study have been deposited in the NCBIGene Expression Omnibus, accession number GSE114363.
1 To whom correspondence should be addressed. E-mail: [email protected].
2 The abbreviations used are: eIF4E, eukaryotic initiation factor 4E; BTF3, basictranscription factor 3; qRT-PCR, quantitative RT-PCR; PBST, PBS with Tween20; FDR, false discovery rate.
systems (15), a mechanism of global control of translation ini-tiation by cap-binding protein sequestration has not yet beendiscovered in plants (8). Arabidopsis thaliana lipoxygenase 2(LOX2) has been shown to interact with eIFiso4E in vitro (16)and A. thaliana basic transcription factor 3 (BTF3) shows inter-action with eIFiso4E in yeast two-hybrid assays (17). However,the biological significance of the observed interaction of theseproteins with eIFiso4E is not known. BTF3 lacks a canonicaleIF4E-binding motif and the proposed eIF4E-binding site ofLOX2 shows poor evolutionary conservation (Fig. S1) (18).
In this study, we describe the discovery of a conserved, plant-specific protein of previously unknown function, conservedbinding of eIF4E 1 (CBE1). CBE1 binds eIF4E in vitro and formscap-binding complexes in an eIF4E-dependent manner in vivo.Plants lacking CBE1 show dysregulation of genes involved incell cycle processes. Based on these observations, we proposeCBE1 and eIF4E may form regulatory messenger ribonucleo-proteins in plants.
Results
CBE1 is a conserved plant cap-binding protein
A previous study by Bush et al. (19) employed MS to identifyproteins from A. thaliana cell culture that are retained on7-methylguanosine Sepharose and are presumably associatedwith cap-binding proteins or complexes. Among the unknownproteins reported, we searched for motifs for interaction withcap-binding proteins. One protein, AT4G01290, appears tohave an N-terminal eIF4E-binding motif. To determinewhether the protein is an evolutionarily conserved eIF4E-bind-ing protein, homologous plant sequences were gathered and aphylogeny was generated (Fig. 1A). The AT4G01290 protein isencoded in genomes across land plants and appears well con-served; however, it is not found outside of plants or in greenalgae. Although AT4G01290 is 991 amino acids in length, noidentified domains are present in the protein, and it does nothave sequence homology to any protein of known function. TheN terminus of AT4G01290 features a very well-conserved
eIF4E-binding motif (Fig. 1B) and based on its features as aunique plant-specific protein with apparent eIF4E-bindingcapacity, we named the protein conserved binding of eIF4E 1(CBE1). The N-terminal domain containing the eIF4E-bindingsite (amino acids 1–230) was selected to determine whether thisprotein is an eIF4E-binding protein.
CBE1 forms cap-binding complexes in vitro
The N-terminal portion of the protein containing the eIF4E-binding motif was expressed as a fusion to GST and purified.GST-CBE11–230 was tested for its ability to bind to the eIF4E-family proteins of A. thaliana, eIF4E, eIFiso4E, and 4EHP (alsoknown as nCBP), by GSH Sepharose pulldown. eIF4E andeIFiso4E were found to bind to GST-CBE11–230 but not to theGST protein control, whereas 4EHP did not bind to GST-CBE11–230 (Fig. 2A). To further test whether the CBE1 proteinmay form an active cap-binding complex, the ability of eIF4Eor eIFiso4E to co-purify GST-CBE11–230 was examined by7-methylguanosine Sepharose pulldown. GST-CBE11–230 wasable to co-purify with the 7-methylguanosine cap analoguebeads in a manner dependent on the presence of eIF4E oreIFiso4E (Fig. 2B), supporting CBE1 as a protein that may forma cap-binding complex with these proteins. To confirm thedirect interaction between CBE1 and cap-binding proteins, theeIF4E-binding site was mutated from (YTRKFLI) to (ATRK-FAA) (GST-CBE1mut1–230) to eliminate the residues essentialto forming a complex with eIF4E, as has previously been shownfor other eIF4E-binding proteins (20, 21). (Underlined lettersindicate residues that were mutated.) The ability to bind eIF4Ewas lost in the GST-CBE1mut1–230 mutant protein (Fig. 2C)further validating CBE1 as a bona fide 4E-binding protein.
The in vitro promiscuity of the eIF4E-binding motifs ofeIF4G and eIFiso4G is known (15, 22). It was our hypothesisthat despite the ability of CBE1 to bind either eIF4E or eIFiso4Ein vitro, it would more likely to form a complex with only eIF4Ein vivo. CBE1 evolved in land plants before eIFiso4E was avail-able, as eIFiso4E is not found in the plant lineage until the
Figure 1. Conserved binding of eIF4E 1 (CBE1) is a conserved plant-specific protein with a predicted eIF4E-binding motif. A, phylogenetic tree ofpredicted CBE1 proteins from representative plant species. The phylogenetic tree was generated using the http://www.phylogeny.fr/ pipeline (39) withalignment by MUSCLE and tree construction by PhyML using 500 bootstrap replicates. (Please note that the JBC is not responsible for the long-term archivingand maintenance of this site or any other third party hosted site.) B, alignment of the N-terminal eIF4E-binding motif (Y(x)4L�) of selected CBE1 proteins in blue(eIF4G and eIFiso4G motifs shown for comparison in green).
appearance of flowering plants (23). Additionally, the eIF4E-binding motif of CBE1 bears slightly more similarity to that ofeIF4G than eIFiso4G. To test whether eIF4E is the preferredbinding partner of CBE1, a mixture of GST-CBE11–230 witheIF4E and eIFiso4E was challenged with excess levels of eithereIF4E or eIFiso4E and subjected to GSH Sepharose pulldown(Fig. 2D). Excess levels of eIF4E led to lower levels of eIFiso4Eco-purification, but excess eIFiso4E did not diminish eIF4Ebinding. These results support eIF4E as the most likely pre-ferred binding partner of CBE1.
CBE1 forms cap-binding complexes in vivo
Although in vitro evidence supports CBE1 as an eIF4E-bind-ing protein, we sought evidence that they form a complex invivo. Cap-binding complexes were co-purified by 7-methyl-guanosine Sepharose beads from seedling lysates of either WTCol0 or cum1 mutant plants, which contain a nonsense muta-tion in the gene encoding eIF4E protein and lack detectable
eIF4E (24). The purified proteins were subjected to MS (Fig. 3)in order to identify eIF4E-dependent cap-binding complexes.eIFiso4F and nuclear cap-binding complex componentsCBP20/CBP80 were identified in cap-binding complex eluatesfrom both Col0 and the eIF4E-lacking cum1 plant lysates. How-ever, eIF4E, eIF4G and CBE1 were found only in the Col0 lysateand were not identified in cap-binding complexes purified fromthe cum1 mutant plants. Based on these observations, webelieve that eIF4E and CBE1 likely form a cap-binding complexin vivo.
CBE1 contributes to plant development and gene regulation
To further investigate the role of CBE1 in plant gene regula-tion, we obtained the T-DNA line cbe1 with an insertion in thesixth exon of the gene, which was confirmed to eliminateexpression of the full-length CBE1 transcript (Fig. 4, A and B).cbe1 plants are viable and robust but show delayed develop-ment relative to WT Col0 plants (Fig. 4C). As CBE1 is encoded
Figure 2. CBE1 forms cap-binding complexes in vitro. A, GST-tag pulldown of recombinant A. thaliana eIF4E, eIFiso4E, or 4EHP in an equimolar mixture withGST control (left) or GST-CBE11–230 (right). Protein mixtures were incubated with GSH Sepharose 4B beads and washed, and bound complexes were eluted withGSH and detected by Western blotting. The eIFiso4E signal extends into eIF4E panel because of sequential probing with anti-eIFiso4E and then anti-eIF4Eantibodies. B, 7-methylguanosine Sepharose bead pulldown of cap-binding complexes. Equimolar mixtures of GST-CBE11–230 with either recombinant A. thali-ana eIF4E or eIFiso4E (as well as GST-CBE11–230 only) were incubated with 7-methylguanosine Sepharose beads and washed, and bound complexes wereeluted with 2� Laemmli sample buffer. Eluted proteins were detected by Western blotting. C, GST-tag pulldown of recombinant A. thaliana eIF4E, in anequimolar mixture with GST-CBE11–230 or GST-CBE1mut1–230 with eIF4E alone as a control. Protein mixtures were incubated with GSH Sepharose 4B beads andwashed; bound complexes were eluted with GSH and detected by Western blotting. D, competition pulldown of recombinant A. thaliana eIF4E and eIFiso4Ewith GST-CBE11–230. Equimolar amounts of GST-CBE11–230 and the two cap-binding proteins were mixed and incubated with GSH Sepharose 4B beads,followed by washing and elution with GSH. GST-CBE11–230– dependent co-purification of cap-binding proteins was directly measured by Bio-Rad stain free gelimaging. The recovery of the respective cap-binding proteins in the pulldown when challenged with 2-fold (��) or 4-fold (���) excess levels of either eIF4Eor eIFiso4E was observed. All experiments were performed three times with similar results.
by a single gene in A. thaliana and has no clear homologues, itappears nonessential, as observed for single gene knockouts ofmost cap-binding complex components (24 –28). As the cbe1T-DNA insertion occurs downstream of the eIF4E-binding site,it is possible a partial N-terminal product with eIF4E-bindingcapacity might be present in the mutant; however, the RNA-Seq data (see below) indicate that the transcript formed is notprocessed correctly and it is unlikely any protein product ismade. The effects of the cbe1 mutation on plant developmentwere examined, along with the cum1 mutant lacking eIF4E anda mutant of eif4g, a T-DNA line lacking full-length EIF4G tran-script (Fig. S2) (28). A delayed flowering time under long daygrowth was observed for cbe1, and this delay in flowering is alsoshared by both the cum1 and eif4g mutants (Fig. 4D). Interest-ingly, the rate of root growth was unaffected in cbe1, whereascum1 and eif4g show slower rates of root elongation than WT(Fig. S3). The presence of eIF4E, as well as its large subunitbinding partner eIF4G, therefore seems necessary for properexecution of the developmental program in A. thaliana.
RNA-Seq of mRNA from Col0 and cbe1 seedlings was per-formed, revealing up-regulation of 230 nuclear-encoded genesand down-regulation of 122 genes in the mutant line (Table S1).Genes overexpressed in the cbe1 mutant have Gene Ontology(GO) term enrichment in cell cycle processes, particularly thoserelated to mitotic plate division (Fig. 5A), whereas down-regu-lated genes show no significantly enriched gene ontology pro-cesses. The G2/M boundary in the cell cycle is an importantcheckpoint in assuring genome integrity and preparing the cellfor division by forming mitotic spindles (29). Previously identi-fied genes with an expression peak at the G2/M boundary (30)show significant enrichment in cbe1 mutant seedlings; 23 out of82 of these genes are up-regulated in the mutant (Fig. 5C).
Overexpression of G2/M-specific genes in cbe1 was con-firmed by testing six representative genes by qRT-PCR in WTand mutant plants as well as eIF4E (cum1) and eIF4G (eif4g)knockout backgrounds (Fig. 5B). Five of the six genes showedsignificant up-regulation in the cbe1 mutant, whereas nonewere affected in the cum1 or eif4g plants. These genes include
Figure 3. CBE1 forms complexes with eIF4E in vivo. Mass spectrometry of native cap-binding complexes isolated from Col0 or cum1 seedlings wasperformed. 7-Methylguanosine Sepharose bead pulldown of seedling lysates was run briefly by SDS-PAGE, and a gel slice containing the protein mixture wasdigested and presented for MS. Numbers shown represent total peptide spectra matching the constituents of cap-binding complexes, with the experimentperformed in triplicate.
Figure 4. cbe1 mutant of A. thaliana. A, site of T-DNA insertion in the sixth exon of the CBE1 coding region. The eIF4E-binding motif is present in the secondexon. B, RT-PCR of 7-day-old WT and cbe1 seedlings grown on MS plates. CBE1 expression is lost in the cbe1 mutant. TUB2 expression serves as a control. C, cbe1mutants show developmental delay relative to WT Col0 plants. Six-week-old plants grown under long day conditions (16 h light, 8 h dark) shown. D, cap-binding complex mutants show delayed flowering time under long day conditions. Error bars represent S.E. (n � 24 –28). Asterisks indicate significantly delayedflowering time in mutant lines compared with Col0 (p value � 0.05, Student’s t test).
the CYCB2.4 and CDKB2.1 genes involved in cyclin-dependentregulation at the G2/M checkpoint (30) and CDC20.2, which isan important component of the mitotic checkpoint complex(31). TPX2 and ATK5, important regulators of mitotic spindleassembly (29, 32), also show increased expression in the cbe1mutant. The results of the qRT-PCR confirm that disruption ofCBE1 activity in A. thaliana leads to overexpression of genesinvolved in mitotic cytokinesis at the G2/M boundary of the cellcycle, suggesting a role in regulation. Leaf epidermal cells wereco-stained with propidium iodide and 4�,6-diamidino-2-phe-nylindole (DAPI) and imaged with a confocal microscope (Fig.S4); however, there were no significant differences in cell mor-phology or density noted between cbe1 and Col0.
Discussion
Whereas mechanisms that regulate translation initiation inother eukaryotic systems such as mammals and yeast are wellunderstood, similar mechanisms have largely not been identi-fied in plants. RNA-binding proteins have been describedwhich regulate the translational state of specific transcripts, butglobal mechanisms for controlling translation remain unclear.In other eukaryotic systems, 4E-BPs control the availability ofeIF4E for translation initiation, or by forming complexes witheIF4E that bind to transcripts to repress translation by blockingrecognition of the mRNA cap by eIF4F. To date, the presence ofsimilar eIF4E-binding proteins in plants with primary roles ingene regulation has not been substantiated.
This report describes a plant-specific protein, CBE1, with anevolutionarily conserved N-terminal motif for binding eIF4E.A. thaliana CBE1 has the capability to bind eIF4E and eIFiso4Eto form cap-binding complexes in vitro and forms binding com-plexes in vivo in an eIF4E-dependent manner. Plants lackingCBE1 protein show dysregulation of cell cycle transcripts, accu-mulating higher levels of mRNA-encoding proteins involved inmitotic processes relative to WT plants.
Although the lipoxygenase LOX2 has previously been dem-onstrated to have the ability to bind eIFiso4E, its potential
eIF4E-binding capacity was only shown in a LacZ activity assay(16). CBE1 represents the first bona fide plant protein describedoutside of eIF4G with strong evidence of direct interaction witheIF4E to form a cap-binding complex that may contribute togene regulation. Outside of the eIF4E-binding motif, CBE1 con-tains no known functional domains and the processes in whichit may be involved are unclear. It has, however, recently beenidentified as a member of the mRNA-bound proteome (33),strengthening the case that it serves a role in gene regulation.
CBE1 appears to be required for proper regulation of mitoticcell cycle transcripts, and mutants lacking eIF4E or eIF4G donot appear to have similar effects on these transcripts. Singledeletions of eIF4E or eIFiso4E have minor effects in A. thali-ana, apparently because of compensation by promiscuousbinding of the alternative available protein, whereas doublemutants are lethal (23, 34). It is unclear, therefore, whethergene regulatory activity of CBE1 is cap-dependent, based onthe observations here. It is also not clear whether CBE1 isinvolved in an upstream process of regulating G2/M specificgenes or whether it acts directly on these transcripts in someway.
Further investigation of the eIF4E-CBE1 complex will berequired to determine the mechanism by which the complexcontributes to specific gene regulation. As CBE1 is a large pro-tein, it could potentially act as a scaffold similarly to eIF4G andeIFiso4G to recruit other effector proteins to bound transcripts.However, as it lacks the HEAT domains of eIF4G family pro-teins that provide interaction with eIF4A and other translationinitiation components, it may have more in common with pro-teins involved in translational repression of targeted tran-scripts. The downstream effects of loss of CBE1 implicate it as apotential regulator of important transcript(s) involved in theG2/M cell cycle checkpoint. Mammalian translation factors areknown to be important regulators of cell cycle progression (35–37). CBE1 may constitute the first plant-specific factor acting ina similar manner.
Figure 5. cbe1 mutants show dysregulation of cell cycle gene expression. A, GO term enrichment of cbe1 mutant up-regulated genes by AmiGO 2 (44). Noenriched GO terms were observed for down-regulated genes in the mutant. B, qRT-PCR of G2/M phase–specific genes identified by sequencing as overex-pressed in cbe1 plants. Gene expression relative to TUB2 was measured in three biological replicates with at least three technical replicates. Error bars representS.E. (n � 3– 6). Asterisks indicate genes with expression significantly varying in the cbe1 mutant compared with Col0 (p value � 0.05, Student’s t test). C, Venndiagram showing overlap of up-regulated genes in cbe1 mutant and G2/M phase–specific genes (30).
A. thaliana T-DNA lines cbe1–1 (WiscDsLoxHs188_10F)and eif4g (SALK_008031) were obtained from the ArabidopsisBiological Resource Center (Ohio State University). The cum1mutant of eIF4E has been described previously (24).
CBE1 phylogeny
Predicted protein sequences from representative plant spe-cies with homology to A. thaliana CBE1 were collected usingPhytozome (38). A phylogenetic tree was generated using thehttp://www.phylogeny.fr/3 pipeline (39) with alignment byMUSCLE and tree construction by PhyML using 500 boot-strap replicates.
CBE1 cloning and expression
A. thaliana CBE1 coding sequence was codon optimizedusing DNAWorks (40) and cloned by overlap PCR. As the full-length protein was prone to high levels of degradation whenexpressed in Escherichia coli, the N-terminal coding portion(amino acid residues 1–230) was subcloned into pGEX-4T-1(GE Healthcare) for expression of GST-tagged construct. GST-CBE11–230 was expressed in BL21(DE3) E. coli and purifiedusing GSH Sepharose 4B (GE Healthcare) and dialyzed in PBS,pH 7.3. Purification of eIF4E, eIFiso4E, and 4EHP were asdescribed previously (22, 41). Inverse PCR with the Q5� Site-Directed Mutagenesis Kit (New England Biolabs) was used tocreate the CBE1 substitution mutation (GST-CBE1mut1–230).The mutation was confirmed by DNA sequencing. Themutant protein was expressed and purified as described forGST-CBE11–230.
Pulldown assays
For GST pulldown assays, A. thaliana eIF4E, eIFiso4E,or 4EHP was mixed with either GST-CBE11–230, GST-CBE1mut1–230, or GST in equimolar ratio in PBS, pH 7.3. Themixtures were added to pre-equilibrated GSH Sepharose 4Bbeads and incubated on ice for 10 min with mixing. The beadswere then washed three times with PBS, pH 7.3, and the proteinwas eluted with 10 mM GSH, 50 mM Tris-HCl, pH 7.6. For7-methylguanosine pulldown assays, GST-CBE11–230 wasmixed with eIF4E or eIFiso4E (or buffer control) in equimolarratio in binding buffer (100 mM HEPES, pH 7.6, 5% glycerol, 50mM KCl, 5 mM EDTA, 0.1% Triton X-100, 5 mM DTT). Themixture (or control) was added to pre-equilibrated 7-methyl-guanosine Sepharose beads (Jena Bioscience) and incubated onice for 20 min with mixing. The beads were then washed fourtimes with buffer, and protein was eluted with 2� LaemmliSample Buffer (Bio-Rad). All pulldown assays were repeated atleast three times with similar results.
Competition assay
Equimolar mixtures of eIF4E, eIFiso4E, and GST-CBE11–230in PBS, pH 7.3, were subjected to competition with 2-fold or
4-fold molar excess of eIF4E or eIFiso4E. The mixtures wereused in a GST pulldown as described above. The input andeluted fractions (10 �l) were separated on a Mini-PROTEAN�TGX Stain-FreeTM gel (Bio-Rad) to directly image the proteinsas per the manufacturer’s instructions using the ChemiDoc MPImager (Bio-Rad) and ImageLab software (Bio-Rad).
Western blotting
Samples were separated by SDS-PAGE on 4 –20% Mini-PROTEAN TGX (Bio-Rad) gel, turbo blotted onto PVDF,blocked with PBST (8 mM Na2HPO4, 0.15 M NaCl, 2 mM
KH2PO4, 3 mM KCl, 0.05% Tween� 20, pH 7.4) containing 5%nonfat dry milk and probed with rabbit antibodies (1:1000 inPBST/milk) to A. thaliana eIF4E (22), A. thaliana eIFiso4E(26), A. thaliana 4EHP (41), or GST (Thermo Fisher Scientific).Horseradish peroxidase–linked secondary antibody (Kirkeg-arrd-Perry, 1/20,000 in PBST/milk) was detected with Super-Signal West Pico or Femto chemiluminescent substrates(Thermo Fisher Scientific). Blots were imaged with a Chemi-Doc MP Imager (Bio-Rad) and ImageLab software (Bio-Rad).
Cap-binding complex MS
Cap-binding complexes were purified from cell lysate of 300mg tissue from Col0 or cum1 12-day-old seedlings grown on 1%sucrose MS plates. The tissue was frozen in liquid nitrogen,ground, and homogenized in binding buffer described above for7-methylguanosine pulldown with addition of Pierce ProteaseInhibitor (Thermo Fisher Scientific). The homogenized lysatewas clarified by centrifugation and added to pre-equilibrated7-methylguanosine Sepharose beads (Jena Bioscience) for over-night incubation at 4 °C with mixing. The beads were washedfour times with buffer and then eluted with 2� Laemmli Sam-ple Buffer (Bio-Rad). The eluate was loaded onto a 4 –20%Mini-PROTEAN TGX (Bio-Rad) gel and briefly subjected toelectrophoresis (about 10 min) and Coomassie Blue stained,and the protein mixture gel band excised for MS. The gel slicewas submitted to The University of Texas at Austin ProteomicsFacility for trypsin digest and LC-MS/MS analysis by OrbiTrapFusion (Thermo Scientific) with spectra visualization by Scaf-fold 4 (Proteome Software). Cutoff for inclusion of spectra wasset at 5% FDR for protein and 0.1% FDR for peptide.
RNA-Seq
Tissue from 7-day-old seedlings grown on 1% sucrose MSplates was frozen in liquid nitrogen and powdered by mortarand pestle, and the RNA was extracted using RNeasy Plant MiniKit (Qiagen). The RNA was treated with DNase I recombinantRNase-free (Roche) and purified using the RNeasy Mini Kit(Qiagen). Poly(A) RNA libraries were constructed andsequenced by the Genome Sequencing and Analysis Facility atThe University of Texas at Austin. Sequencing data weremapped with TopHat to the Arabidopsis thaliana genomerelease 10 (42), and genes with significantly altered expressionwere identified with CuffDiff (43). Differentially regulatedgenes were defined as having a 2-fold change in gene expressionand an FDR-adjusted p value (q-value) �0.05. Gene ontologyterm enrichment analysis was performed with AmiGO 2 (44).
3 Please note that the JBC is not responsible for the long-term archiving andmaintenance of this site or any other third party hosted site.
RNA sequence data are available in the Gene Expression Omni-bus (GEO) GSE114363.
qRT-PCR
Quantitative real-time PCR was performed on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) using PowerUpSYBR Green (Applied Biosystems) under standard conditions.RNA was extracted from 7-day-old seedlings as described forRNA-Seq. cDNA was generated with oligo(dT) and SuperscriptIV Reverse Polymerase (Thermo Fisher Scientific). qRT-PCRprimers were designed using Primer3 (45) and are listed inTable S2. TUB2 primers were described previously (46).
Propidium iodide staining
Leaves from 21-day-old A. thaliana Col0 or cbe1 seedlingswere floated in 10 �g/ml propidium iodide and 1 �g/ml 4�,6-diamidino-2-phenylindole (DAPI) in water for 10 min in thedark, then mounted on slides. Confocal microscopy (20�, 514nm laser) was performed at the Center for Biomedical ResearchSupport at the University of Texas at Austin with a Zeiss LSM710 confocal microscope (ZEISS).
Author contributions—R. M. P. conceptualization; R. M. P. andJ. C. H. L. data curation; R. M. P., J. C. H. L., J. R. J. T., S.-H. Y.,G. S. C., and K. S. B. formal analysis; R. M. P., S.-H. Y., G. S. C., andK. S. B. supervision; R. M. P., J. C. H. L., and K. S. B. validation;R. M. P., J. C. H. L., J. R. J. T., S.-H. Y., G. S. C., and K. S. B. investiga-tion; R. M. P. visualization; R. M. P., J. C. H. L., S.-H. Y., G. S. C., andK. S. B. methodology; R. M. P. and K. S. B. writing-original draft;R. M. P., S.-H. Y., G. S. C., and K. S. B. writing-review and editing;K. S. B. resources; K. S. B. funding acquisition; K. S. B. projectadministration.
Acknowledgments—We thank Dr. Andrew Lellis and Anna Webb(Center for Biomedical Research Support) for assistance with themicroscopy.
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